Lymphocyte-endothelial cell interactions are mediated in part by multiple lymphocyte surface adhesion/activation molecules and their cognate ligands. We investigated the surface localization of several of these molecules implicated in T cell adhesion and transen- dothelial migration mechanisms to determine if spatial regulation of their distribution contributes to these processes. T lymphocyte suspensions were stained to define distribution, ability to be aggregated into energy- dependent caps, and potential cocapping of several adhesion structures. CD2, CD44, L-selectin (LAM-1, LECCAM-1), and CDlla/CD18 (LFA-1) exhibited uniform distribution on the T cell surface by direct immunofluorescence but formed caps in an energy- dependent, and therefore cytoskeletally driven, manner when examined by indirect immunofluorescence. CD2 was shown to syn-cap (unidirectionally cocap) with GD44 and CDlla/CD18 (LFA-1), an observation potentially related to functional cooperation among these molecules in T cell activation. T cells were also added to endothelial cell monolayers to assess, in a physiologically relevant context, potential surface molecule reorganization. Lymphocytes co-cultured with human umbilical vein endothelial cells (HUVEC) underwent a profound shape change, from essentially round cells to polarized cells bearing pseudopodia. Immunofluorescent localization of T cell adhesion/activation molecules using confocal microscopy revealed the redistribution of CD2, CD44, and L-selectin to the pseudopod. In contrast, CDlla/CD18 remained globally distributed on the cell surface, even in severely deformed cells. Both lymphocyte shape change and membrane molecule redistribution appear to be cell-cell contact-dependent phenomena requiring intact, viable endothelial cells. Mechanisms that control these events may be critical to lymphocyte recirculation and inflammation. J. Leukoc. Biol. 53: 1–10; 1993.